It is well established that diabetes leads to an increased risk for the development of heart failure independent of other risk factors such as hypertension and ischemic heart disease; however, there is no consensus as to the mechanisms involved or the most appropriate treatment strategies. High levels of glucose activate the hexosamine biosynthesis pathway (HBP) and increase the levels of N-acetylglucosamine (O-GlcNAc) on cytoplasmic and nuclear proteins. Our preliminary data suggest that the HBP and protein O-GlcNAcylation contribute to diabetes-induced myocyte dysfunction. Indeed, increased flux through HBP may represent a critical link between metabolic dysfunction and impaired cardiac function following the development of diabetes. Therefore, the overall hypothesis of this proposal is that the dysregulation of myocardial carbohydrate and fatty acid metabolism in Type-2 diabetes leads to increased flux through the HBP and results in increased O-GlcNAcylation and altered behavior of proteins involved in regulating normal cardiomyocyte function. Consequently in hearts from normal, insulin resistant and diabetic rats we will: 1) determine the impact of altered carbohydrate and fatty acid metabolism on flux through the HBP, phosphorylation of the MAPK and the PI3K/Akt/GSK pathways; 2) determine whether increased HBP flux and protein O-GlcNAcylation in Type-2 diabetes contributes to impaired cardiac function including altered response to a-adrenergic stimulation and activation of pro-apoptotic and inhibition of prosurvival components of the MAPK and PI3K/Akt/GSK pathways; 3) Identify specific proteins that are modified including those involved in a-adrenergic signaling and the MAPK and the PI3K/Akt/GSK pathways that are integral to the adverse effects of diabetes on the heart. These studies bring together 13C-NMR spectroscopy with cellular, molecular and proteomic techniques to evaluate the interactions between glucose and fatty acid utilization, the HBP, protein O-GlcNAcylation and cardiac function in hearts from normal and diabetic animals. We will use the Zucker diabetic fatty rat, a model of Type 2 diabetes, which includes insulin resistance, hyperglycemia and obesity. The successful outcome of these studies will provide the foundation for an understanding of the molecular mechanisms underlying the impact of Type 2 diabetes and insulin resistance on cardiac dysfunction. This will facilitate the development of novel therapeutic interventions, designed to close the gap in mortality and morbidity between diabetic and non-diabetic patients with heart disease. [unreadable] [unreadable]